Materials and methods relating to the diagnosis and treatment of pre-eclampsia and diabetes

ABSTRACT

The present invention relates to materials and methods for the diagnosis and treatment of pre-eclampsia, and more particularly to the role of P-type inositolphosphoglycans (IPGs) in the occurrence of pre-eclampsia. Methods of diagnosing pre-eclampsia by determining the level of P-type IPGs and uses of antagonists of P-type IPGs in the treatment of pre-eclampsia are disclosed, together with a method for screening for P-type IPG antagonists.

This Application is a 35 USC 371 of PCT/GB97/02523, filed Sep. 11, 1997.

1. Field of the Invention

The present invention relates to materials and methods for the diagnosisand treatment of pre-eclampsia and diabetes, and more particularly tothe role of P-type inositolphosphoglycans (IPGs) in the occurrence ofpre-eclampsia, methods of diagnosing pre-eclampsia and uses ofantagonists of P-type IPGs in the treatment of pre-eclampsia.

2. Background of the Invention

Pre-eclampsia is a placental disease [1] characterised by insufficiencyof the uteroplacental circulation [2], and which affects 10-12% of allpregnancies and is a major factor in the perinatal mortality rate. Thereis evidence that one or more placentally-derived factors are releasedinto the maternal circulation which either directly or indirectly causematernal endothelial dysfunction and ensuing maternal problems withactivation of the clotting system increased vascular permeability andischaemia in maternal organs secondary to vasoconstriction [3].

SUMMARY OF THE INVENTION

The present invention arises from investigations to determine whetherthere is a correlation between pre-eclampsia and its degree of severityand the profile of inositol phosphoglycans (IPGs) in the pre-eclampticsubjects and their normal age and parity-matched controls. In order togain information on the significance of the disordered carbohydratemetabolism in the placenta in pre-eclampsia, as previously revealed bythe massive increase in glycogen accumulation [4], comparison has beenmade with diabetic pregnant women in which placental glycogenaccumulation is also a prominent feature [4,5], although not accompaniedby the same degree of the life-threatening sequelae of pre-eclampsia.

Accordingly, in a first aspect, the present invention provides the useof a P-type inositolphosphoglycan (IPG) antagonist in the preparation ofa medicament for the treatment of pre-eclampsia.

In further aspect, the present invention provides a method of treatingpre-eclampsia in a patient, the method comprising administering atherapeutically effective amount of a P-type IPG antagonist to apatient.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a P-type antagonist in combination with apharmaceutically acceptable carrier.

P-type IPGs and method for isolating them from human tissue aredescribed below. This in turn allows those of ordinary skill in the artto prepare P-type IPG antagonists.

In the present invention, “P-type IPG antagonists” includes substanceswhich have one or more of the following properties:

(a) inhibiting the release of P-type IPG from placenta;

(b) reducing the levels of placenta derived P-type IPG via an IPGbinding substance (e.g. an antibody or a specific binding protein)against the placental derived TPG; and/or,

(c) reducing the effects of placenta derived P-type IPG.

In a further aspect, the present invention provides a method ofscreening for P-type IPG antagonists, the method comprising:

(a) contacting a candidate antagonist and a P-type IPG in an assay for abiological property of the P-type IPG under conditions in which theP-type IPG and the candidate antagonist can compete;

(b) measuring the biological property of the P-type IPG; and,

(c) selecting candidate antagonists which reduce the biological activityof the P-type IPG.

Some of the biological properties of P-type IPGs and assays to determinethese properties that can be used in the above screening method are setout in the description below. The techniques of combinatorial chemistryare particularly suited to the production of large numbers of syntheticcandidate antagonists, which can be screened for activity in the abovemethod.

The particular emphasis placed upon the determination of the output ofIPGs in both pre-eclamptic and diabetic pregnant women relates to theknown fundamental importance of the class of compound in regulation keysites in metabolic pathways, resulting, in different tissues, in thedirection of carbohydrates towards oxidation and glycogen synthesis inthe case of the IPG P-type, or towards lipogenesis in the case of theIPG A-type; this regulation being both organ and inter-organ related [6,9].

In copending applications claiming priority from GB-A-9618934.5, wereport on the urinary content of IPGs in diabetic patients, from whichevidence has been adduced for a critical role of altered inositolphosphoglycan profiles in relation to parameters linked to syndrome X,such as insulin resistance, obesity and high blood pressure. There aresimilarities between the metabolic changes in pre-eclampsia and syndromeX [10].

The results of our investigations described below indicate thefollowing:

(a) The 24 hour output of IPG P-type in urine in pre-eclamptic women issignificantly higher (2- to 3-fold) than in pregnant control subjectsmatched for age, parity and stage of gestation.

(b) Diabetic pregnant women do not show any significant change inurinary output of IPG P-type relative to pregnant control subjectsmatched for age, parity and gestational stage.

(c) Pregnancy itself is associated with an increased urinary output ofIPG P-type relative to non-pregnant controls matched for age.

(d) No significant changes were found in the daily output of IPG A-typein pre-eclamptic or diabetic groups, with the exception of an increasein the IPG A-type in the pre-eclamptic group when the results wereexpressed as units per mmole creatinine.

(e) Urinary excretion of IPG P-type correlated with markers of theseverity of pre-eclampsia, plasma alanine aspartate transaminase, degreeof proteinuria and with platelet counts.

(f) Human placenta contained very high concentrations of IPG-P type,some 100×greater than either human or rat liver. It also appears tocontain an inhibitor of IPG-P-ype activity as evidenced by a calculateddecrease in activity when increasing the volume of the same preparationtested in the PDH phosphatase system (FIG. 7). Pre-eclamptic placentacontains approximately twice as much IPG-P type as does placenta fromnormal pregnant subjects. The IPG-A (pH 1.3 fraction) isolated fromplacenta showed no activity when tested for its ability to stimulatelipogenesis in rat adipocytes.

(g) Evidence has been found suggesting that the use of contraceptivepills may be related to an increase in IPG P-type in urine of normalwomen. (5 values only in each group).

(h) After storage for 10 months at −8° C., urine from pre-eclampticwomen showed increased P-type activity (FIG. 8A) indicating that theurine initially contained a labile inhibitor. The yield of the IPGA-type isolated from the same urines decreased in activity (FIG. 8B).

(i) Significant differences were found between the ratios of IPG P-typeand IPG A-type in non-pregnant women and normal male subjects; while theIPG P-type was similar in both groups, the IPG A-type was 5- to 6-foldhigher in women.

(j) There is a 2.7 fold increase in IPG P-type in the urine ofpre-eclamptic women, compared to normal pregnant subjects. There is a2.7 fold increase in placenta-derived P-type mediators frompre-eclamptic women compared to normal pregnant subjects (See Table 5).

(k) The high urinary excretion IPG P-type in pre-eclampsia reflects highplacental and circulating levels and is directly related to theaccumulation of glycogen in the placenta in this condition, because IPGP-type activates glycogen synthase phosphatase.

(l) The concentration of P-type mediators in the urine of pre-eclampticwomen returns baseline in post natal sample, (See FIG. 6) confirms thatthe source of the relevant P-type mediator in pre-eclamptic women is theplacenta.

(m) A high circulating level of IPG P-type originating in the placentamay have paradrine effects, eg: in stimulating other endocrine glands,and/or affecting endothelial cells which could contribute to thepathogenesis of the pre-eclampsia syndrome.

The present invention provides, inter alia, a therapeutic treatment ofpre-eclampsia:

(1) to inhibit the release of P-type mediator from placenta;

(2) to reduce the levels of placenta derived P-type IPG via antibodyagainst the placental derived IPG;

(3) to reduce the effects of placenta derived P-type IPG via P-typeantagonist.

The substance can be administered as the sole active substance, or as anadjunct to other forms of treatment. Because of their small molecularweight and heat and acid stability, IPGs should be suitable for oraladministration, but other forms of administration are also contemplated.In the case of antibodies, or other proteins or substances which may notbe suitable for oral administration, other methods such as parenteraladministration may be used. Antibodies for administration are preferablyhuman or “humanised” according to known techniques. This is discussedfurther below.

The invention also contemplates measurement of P-type IPG in blood orurine as a diagnostic for pre-eclampsia. Thus, in a further aspect, thepresent invention provides a method of diagnosing pre-eclampsia in apatient, the method comprising determining the level of P-type IPGs in abiological sample: obtained from the patient. Thus, a diagnosis can thenbe made by correlating this level with known levels of the P-type IPGS.

In one embodiment, the method comprises the steps of:

(a) contacting a biological sample obtained from the patient with asolid support having immobilised thereon binding agent having bindingsites specific for one or more P-type IPGs;

(b) contacting the solid support with a labelled developing agentcapable of binding to unoccupied binding sites, bound P-type IPGs oroccupied binding sites; and,

(c) detecting the label of the developing agent specifically binding instep (b) to obtain a value representative of the level of the P-typeIPGs in the sample.

As set out below, in this aspect of the invention, the level of theP-type IPGs can be further confirmed using a marker which correlateswith the level of the P-type IPGs.

The present invention will now be described by way of example and notlimitation with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A, B) individual values for the concentration of IPGP-type (units/mmole creatine) in the urine of pregnant women in thepre-eclamptic group, diabetic group, in their respective matched normalpregnancy groups and in non-pregnant women, and (C, D) individual valuesfor the concentration of IPG A-type (units/mmole creatine) in the urineof pregnant women in the pre-eclamptic group, diabetic group, and theirrespective matched normal pregnancy groups and non-pregnant women.

FIGS. 2A, B shows the effect of the contraceptive pill on urinary outputof IPG P-type and IPG A-type in non-pregnant control subjects.

FIGS. 3A, B shows the relationship between P-type and stage of gestationat which samples were collected from groups of pre-eclamptic, diabeticand matched control pregnant women.

FIG. 4 shows (A) the relationship between urine IPG P-type and proteinin urine in pre-eclampsia, (B) the relationship between urine IPG P-typeand elevated plasma levels of alanine-aspartate transaminase, and (C)the relationship between urine IPG P-type and platelet counts inpre-eclampsia.

FIG. 5 shows (A) and (B) the concentration of IPG P-type in urine inpre-eclampsia ante-natal and post-natal samples, and (C) urine volumesin pre-eclampsia ante-natal and post-natal samples.

FIG. 6 shows a scheme setting out factors regulating placental glycogenmetabolism in pre-eclampsia and diabetes.

FIG. 7 shows the effect of weight of tissue and volume used in assay onthe estimation of P-type IPG in placenta of normal subjects.

FIGS. 8A, B show evidence for an unstable inhibitor of IPG-P type in theurine of pregnant women, both in normal and pre-eclamptic subjects, andthe effect of storage of urine samples for 10 months at −80° C. on theP-and A-type IPG activity.

FIGS. 9 and 10 shows the relationship between the yield of P-type IPGand the weight of placental tissue extracted in normal and pre-eclampticsubjects.

DETAILED DESCRIPTION OF THE INVENTION

IPGs

Studies have shown that A-type mediators modulate the activity of anumber of insulin-dependent enzymes such as cAMP dependent proteinkinase (inhibits), adenylate cyclase (inhibits) and cAMPphospho-diesterases (stimulates). In contrast, P-type mediators modulatethe activity of insulin-dependent enzymes such as pyruvate dehydrogenasephosphatase (stimulates) and glycogen synthase phosphatase (stimulates).The A-type mediators mimic the lipogenic activity of insulin onadipocytes, whereas the P-type mediators mimic the glycogenic activityof insulin on muscle. Both A-and P-type mediators are mitogenic whenadded to fibroblasts in serum free media. The ability of the mediatorsto stimulate fibroblast proliferation is enhanced if the cells aretransfected with the EGF-receptor. A-type mediators can stimulate cellproliferation in the chick cochleovestibular ganglia.

Soluble IPG fractions having A-type and P-type activity have beenobtained from a variety of animal tissues including rat tissues (liver,kidney, muscle brain, adipose, heart, placenta) and bovine liver. A- andP-type IPG biological activity has also been detected in human liver andplacenta, malaria parasitized RBC and mycobacteria. The ability of ananti-inositolglycan antibody to inhibit insulin action on humanplacental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPGaction on rat diaphragm and chick ganglia suggests cross-speciesconservation of many structural features. However, it is important tonote that although the prior art includes reports of A- and P-type IPGactivity in some biological fractions, the purification orcharacterisation of the agents responsible for the activity is notdisclosed.

In our co-pending patent applications claiming priority fromGB-A-9618930.3 and GB-A-9618929.5, we have described the isolation andcharacterisation of P-type and A-type IPGs.

A-type substances are cyclitol-containing carbohydrates, also containingZn²⁺ ion and optionally phosphate and having the properties ofregulating lipogenic activity and inhibiting cAMP dependent proteinkinase. They may also inhibit adenylate cyclase, be mitogenic when addedto EGF-transfected fibroblasts in serum free medium, and stimulatelipogenesis in adipocytes.

P-type substances are cyclitol-containing carbohydrates, also containingMn²⁺ and/or Zn⁺ ions and optionally phosphate and having the propertiesof regulating glycogen metabolism and activating pyruvate dehydrogenasephosphatase. They may also stimulate the activity of glycogen synthasephosphatase, be mitogenic when added to fibroblasts in serum freemedium, and stimulate pyruvate dehydrogenase phosphatase.

The A- and P-type substances were also found to have the followingproperties:

1. Migrates near the origin in descending paper chromatography using4/1/1 butanol/ethanol/water as a solvent.

2. The substances contains phosphate which is directly related toactivity.

3. The free GPI precursors are resistant to cleavage by GPI-PLC.

4. They are bound on Dowex AG50 (H+) cation exchange resin.

5. They are bound on an AG3A anion exchange resin.

6. The activity is resistant to pronase.

7. They are detected using a Diones chromatography system.

8. The P-type substance is partially retained on C-18 affinity resin.

The A- and P-type substances may be obtained from human liver orplacenta by:

(a) making an extract by heat and acid treatment of a liver homogenate,the homogenate being processed from tissue immediately frozen in liquidnitrogen;

(b) after centrifugation and charcoal treatment, allowing the resultingsolution to interact overnight with an AG1-X8 (formate form) anionexchange resin;

(c) collecting a fraction having A-type IPG activity obtained by elutingthe column with 50 mM HCl, or a fraction having P-type IPG activityobtained by eluting the column with 10 mM HCl;

(d) neutralising to pH 4 (not to exceed pH 7.8) and lyophilising thefraction to isolate the substance.

(e) descending paper chromatography using 4/1/1 butanol/ethanol/water assolvent.

(f) purification using high-voltage paper electrophoresis inpyridine/acetic acid/water.

(g) purification using Dionex anion exchange chromatography orpurification and isolation using Vydac 301 PLX575 HPLC chromatography.

More details of the methods for obtaining these IPGs are provided in thesaid patent applications, the contents of which are incorporated hereinby reference.

Antagonists

As mentioned above, antagonists of P-type activity, either naturallyoccurring or synthetic, inlcude substances which have one or more of thefollowing properties:

(a) substances capable of inhibiting release of P-type mediator fromplacenta;

(b) substances capable of reducing the levels of placenta derived P-typeIPG via an IPG binding substance (e.g. an antibody or specific bindingprotein) against the placental derived IPG; and/or,

(c) substances capable of reducing the effects of placenta derivedP-type IPG.

In one embodiment, the IPG antagonists are specific binding proteins.Naturally occurring specific binding proteins can be obtained byscreening biological samples for proteins that bind to IPGs.

In a further embodiment, the antagonists are antibodies. The productionof polyclonal and monoclonal antibodies is well established in the art,and exemplary protocols are set out in the examples below. Monoclonalantibodies can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP-A-184187, GB-A-2188638or EP-A-239400. A hybridoma producing a monoclonal antibody may besubject to genetic mutation or other changes, which may or may not alterthe binding specificity of antibodies produced.

Antibodies may be obtained using techniques which are standard in theart. Methods of producing antibodies include immunising a mammal (e.g.mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al, Nature, 357:80-82, 1992). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide anantibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see W092/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith any of the proteins (or fragments) , or may be one constructedusing sequences obtained from an organism which has been exposed to theantigen of interest.

Antibodies according to the present invention may be modified in anumber of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or otherbinding partner are the Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′) 2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

Humanised antibodies in which CDRs from a non-human source are graftedonto human framework regions, typically with the alteration of some ofthe framework amino acid residues, to provide antibodies which are lessimmunogenic than the parent non-human antibodies, are also includedwithin the present invention.

A hybridoma producing a monoclonal antibody according to the presentinvention may be subject to genetic mutation or other changes. It willfurther be understood by those skilled in the art that a monoclonalantibody can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP-A-184187, GB-A-2188638or EP-A-0239400. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

Hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells, eukaryotic or prokaryotic, containing nucleic acid encodingantibodies (including antibody fragments) and capable of theirexpression. The invention also provides methods of production of theantibodies including growing a cell capable of producing the antibodyunder conditions in which the antibody is produced, and preferablysecreted.

The antibodies described above may also be employed in the diagnosticaspects of the invention by tagging them with a label or reportermolecule which can directly or indirectly generate detectable, andpreferably measurable, signals. The linkage of reporter molecules may bedirectly or indirectly, covalently, e.g. via a peptide bond ornon-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenizidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

In a further embodiment, the IPG antagonists are synthetic compounds.These may be produced by conventional chemical techniques or usingcombinatorial chemistry, and then screened for IPG antagonist activity.These compounds may be useful in themselves or may be used in the designof mimetics, providing candidate lead compounds for development aspharmaceuticals. Synthetic compounds might be desirable where they arecomparatively easy to synthesize or where they have properties that makethem suitable for administration as pharmaceuticals, e.g. anatgonistwhich are peptides may be unsuitable active agents for oral compositionsif they are degraded by proteases in the alimentary canal. Mimeticdesign, synthesis and testing is generally used to avoid randomlyscreening large number of molecules for a target property.

Production of Monoclonal Antibodies

Inositolphosphoglycan (IPG) purified from rat liver by sequential thinlayer chromatography (TLC) was used to immunize New Zealand rabbits andBalb/c mice by using conventional procedures.

After immunisation, monoclonal antibodies were prepared using theapproach of fusion of mouse splenocytes (5×10⁶ cells/ml) with mutantmyeloma cells (10⁶ cells/ml). The myeloma cell lines used were thoselacking hypoxanthine-guanine phosphoribasyl transferase. The screeningmethod of hybridoma cells was based on a non-competitive solid-phaseenzyme immunoassay in which the antigen (IPG) was immobilised on a solidphase. Culture supernatant were added and positive hybridoma cells wereselected.

A single cell cloning was made by limiting dilution. Hybridomas forthree monoclonal antibodies (2D1, 5HG and 2P7) were selected. Allmonoclonal antibodies were determined to be IgM using a EK-5050 kit(Hyclone).

In order to test that all monoclonal antibodies recognised IPGs, anon-competitive solid-phase enzyme immunoassay was used. F96 PolysorpNunc-Immuno Plates are used for the assay. The polysorp surface isrecommended for assays where certain antigens are immobilised.

The immobilised antigen (IPG) diluted to 1:800 captured the monoclonalantibody from tissue culture supernatant, ascitic fluid, and when thepurified monoclonal antibody was used.

The detection method used an anti-mouse IgM, biotinylated whole antibody(from goat) and a streptavidin-biotinylated horseradish peroxidasecomplex (Amersham), ABTS and buffer for ABTS (Boehringer Mannheim).

The same immunoassay was used to evaluate the polyclonal antibody. Inthis assay, the detection method employed an anti-rabbit Ig,biotinylated species—specific whole antibody (from donkey).

The antibodies can be purified using the following method. Fast ProteinLiquid Chromatography (Pharmacia FPLC system) with a gradient programmerGP-250 Plus and high precision pump P-500 was used in order to purify apolyclonal IPG antibody.

A HiTrap protein A affinity column was used for purification ofpolyclonal IPG from rabbit serum, Protein quantitation was made using aMicro BCA protein assay reagent kit (Pierce).

Monoclonal IgM antibodies were purified in two steps. Ammonium sulfateprecipitation was the method chosen as a first step. Tissue culturesupernatant was treated with ammonium sulfate (50% saturation). Pelletdiluted in PBS was transferred to dialysis tubing before the secondstep.

Since ammonium sulfate precipitation is not suitable for a single steppurification, it was followed by gel filtration chromatography-antibodysolution in PBS run into a Pharmacia Sepharose 4B column. Proteinquantitation was made reading the absorbance at 220-280 nm in aPerkin-Elmer lambda 2 UV/VIS spectrophotometer.

Protocol for Sandwich ELISA

The protocol below sets out an indirect, non-competitive, solid-phaseenzyme immunoassay (sandwich ELISA) for the quantification ofinositolphosphoglycans (IPG) in biological fluids, such as human serum.

In the assay, monoclonal IgM antibodies are immobilised on a solidphase. Tissue culture supernatant, ascitic fluid from mice with aperitoneal tumour induced by injecting hybridoma cells into theperitoneum and purified monoclonal antibody have been used in theimmunoassay. F96 Maxisorp Nunc-Immuno plates were used for these assays.Maxisorp surface is recommended where proteins, specially glycoproteinssuch as antibodies, are bound to the plastic.

The immobilised antibody captures the antigen from the test sample(human serum or IPG used like a positive control).

A bridging antibody (a purified polyclonal IPG antibody from rabbit) isneeded to link the anti-antibody biotinylated to the antigen.

The detection method employs an anti-rabbit Ig, biotinylatedspecies-specific whole antibody (from donkey) and asureptavidin-biotinylated horseradish peroxidase complex (Amersham),ABTS and buffer for ABTS (Boehringer Mannheim).

The ELISA assay can be carried out as follows:

1. Add 100 μl/well in all the steps.

2. Add monoclonal antibody diluted 1:100 in PBS in a F96 MaxisorpNunc-Immuno plate. Incubate at least 2 days at 4° C.

3. Wash with PBS three times.

4. Add a blocking reagent for ELISA (Boehringer Mannheim) in distilledwater (1:9) 2 hours at room temperature.

5. Wash with PBS-Tween 20 (0, 1%) three times.

6. Add a purified polyclonal antibody (diluted 1:100 in PBS), overnightat 4° C.

7. Wash with PBS-Tween 20 (0.1%) three times.

8. Add an anti-rabbit Ig, biotinylated species-specific whole antibody(from donkey) (Amersham) diluted 1:1000 in PBS, 1 h 30 min at roomtemperature.

9. Wash with PBS-Tween 20 (0.1%) three times.

10. Add a streptavidin-biotinylated horseradish peroxidase complex(Amersham) diluted 1:500 in PBS, 1 h 30 min at room temperature.

11. Wash with PBS three times.

12. Add 2.2-Azino-di-(3-ethylbenzthiazoline sulfonate (6)) diammoniumsalt crystals (ABTS) (Boehringer Mannheim) to buffer for ABTS (BM) :Buffer for ABTS is added to distilled water (1:9 v/v). 1 mg of ABTS isadded to 1 ml of diluted buffer for ABTS.

13. Read the absorbance in a Multiscan Plus P 2.01 using a 405 mm filterin 5-15 min.

Pharmaceutical Compositions

The antagonists of the invention can be formulated in pharmaceuticalcompositions. These compositions may comprise, in addition to one ormore of the P-type antagonists, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material may depend on the route ofadministration, e.g. oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Whether it is a polypeptide, antibody, peptide, small molecule or otherpharmaceutically useful compound according to the present invention thatis to be given to an individual, administration is preferably in a“prophylactically effective amount” or a “therapeutically effectiveamount” (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Diagnostic Methods

Methods for determining the concentration of analytes in biologicalsamples from individuals are well known in the art and can be employedin the context of the present invention to determine whether anindividual has an elevated level of P-type IPGs, and so has or is atrisk from pre-eclampsia. The purpose of such analysis may be used fordiagnosis or prognosis to assist a physician in determining the severityor likely course of the pre-eclampsia and/or to optimise treatment ofit. Examples of diagnostic methods are described in the experimentalsection below.

Preferred diagnostic methods rely on the detection of P-type IPGs, anelevated level of which was found to be associated with pre-eclampsia.The methods can employ biological samples such as blood, serum, tissuesamples (especially placenta), or urine.

The assay methods for determining the concentration of P-type IPGstypically employ a binding agent having binding sites capable ofspecifically binding to one or more of the P-type IPGs in preference toother molecules. Examples of binding agents include antibodies,receptors and other molecules capable of specifically binding P-typeIPGs. Conveniently, the binding agent is immobilised on solid support,e.g. at a defined location, to make it easy to manipulate during theassay.

The sample is generally contacted with a binding agent under appropriateconditions so that P-type IPGs present in the sample can bind to thebinding agent. The fractional occupancy of the binding sites of thebinding agent can then be determined using a developing agent or agents.Typically, the developing agents are labelled (e.g. with radioactive,fluorescent or enzyme labels) so that they can be detected usingtechniques well known in the art. Thus, radioactive labels can bedetected using a scintillation counter or other radiation countingdevice, fluorescent labels using a laser and confocal microscope, andenzyme labels by the action of an enzyme label on a substrate, typicallyto produce a colour change. The developing agent can be used in acompetitive method in which the developing agent competes with theanalyte (P-type IPG) for occupied binding sites of the binding agent, ornon-competitive method, in which the labelled developing agent bindsanalyte bound by the binding agent or to occupied binding sites. Bothmethods provide an indication of the number of the binding sitesoccupied by the analyte, and hence the concentration of the analyte inthe sample, e.g. by comparison with standards obtained using samplescontaining known concentrations of the analyte.

EXPERIMENTAL DESCRIPTION A. Experimental

1. Assay of IPG A-type and IPG P-type Activity

The activity of P- and A-type IPGs in urine and placental extracts werestudied using specific bioassay procedures. IPG P-type was determinedusing the activation of PDH phosphatase [11]. The PDH complex and PDHphosphatase (metal-dependent form) were prepared from beef heart asdescribed by Lilley et al. [11] and the assay of the activation of thephosphatase was performed by the spectrophotometric variant of thetwo-stage system described by these authors. This assay is considered tobe a characteristic feature of IPG P-type (see Larner et al. [12]) . IPGA-type was determined by the stimulation of lipogenesis as measured bythe incorporation of [U¹⁴C] glucose into the lipids of adipocytesisolated from epididymal fat pads by the method of Rodbell [13]. A highdegree of specificity for IPG A-type was found for this bioassay.

A straight line relationship between added IPGs and the stimulation ofPDH phosphatase activity (IPG P-type) and lipogenesis in intactadipocytes (IPG A-type) was obtained; this relationship held at least upto a stimulation of +250%. These observations provided a basis for aunit to be defined and used for the purpose of comparison of yields ofIPGs from different tissues and urine samples. Linearity between IPGadded and the percentage change in response, has been observed by others(see Lilley et al. [11] and Newman et al. [14]), although Asplin et al.[15] did not show linearity in their study on IPGs in human urine fromnormal and diabetic subjects, an effect which was particularly markedwith the IPG A-type (pH 1.3 fraction).

2. Extraction of IPG P-type and IPG A-type from Urine

Urines were extracted as described by Asplin et. al. [15]. The finalfractions were freeze dried and stored at −20° C. For use, the IPGfractions were resuspended in water, immediately before assay, so that10 μl of redissolved IPG corresponded to 10 ml urine.

In view of the possibility that high, and varying, amounts of IPGs mightbe excreted in the different groups of pregnant and pre-eclampticsubjects, and in order to ensure that the capacity of the resin was wellin excess of the load applied, preliminary test runs were made todetermine the optimal ratio of resin to starting urine volume. Linearityof recovery was obtained up to 100 ml urine per 18 g resin. In thepresent study, the ratio of 30 ml urine to 18 g resin was maintained toallow for variation in IPG content.

3. Preparation of Placenta

In preliminary studies two normal placentae were obtained and treated asfollows:

(i) The first was collected within 40 minutes of delivery and samples ofthe tissue were freeze-clamped and stored and transported in solid CO₂.

(ii) The second placenta was collected an estimated 30 minutespost-delivery and a 15 g sample freeze-clamped immediately. Theremainder was divided into two, one half stood at room temperature andthe other stored in ice. 10 g samples from each of these halves wereremoved after 1, 3 and 5 hours, freeze-clamped and treated as above.

These samples were stored at −80° C. until extracted.

4. Extraction of Inositol Phosphoalvcans from Placenta

The extraction procedure involved pulverising the frozen tissue (5 g)under liquid nitrogen and then extracting with 50 mM formic acid at 100°C. for 3 minutes. The supernatant fraction, after centrifugation, wastreated with charcoal (10 mg/ml) and again centrifuged. The charcoalsupernatant was passed through a Millipore filter, diluted 5-fold withwater and then brought to pH 6 with ammonia. After centrifugation, theextract was added to 15 g AG1-X8 and stood overnight at 0° C. The resinwas then transferred to columns and washed with 40 ml water followed by40 ml of HCl, pH3. IPG P-type was eluted with 100 ml of HCl, pH 2.0, andIPG A-type with 100 ml HCl, pH 1.3. The extracts were brought to pH 4with ammonia and rotary evaporated to about 5 ml before beingtransferred to smaller tubes and freeze dried. It was stored in thisstate at −20° C. until used. This extraction procedure was based on thatdescribed by Nestler et. al. [16].

The wide variations in inositol phosphoglycan content of differenttissues [91 prompted the preliminary examination of:

(i) the optimal weight of placental tissue relative to the weight ofresin to be used in the isolation and separation of IPG P-type and IPGA-type and

(ii) the amount of the isolated fractions to be used in the bioassaysystems to ensure that the assays fell within the linear portion ofdose-response curve.

It was established that, using a column containing 18 ml resin, maximumIPG-P type activity was recovered when placenta samples were less than0.3 g (FIGS. 9 and 10) and that the final assay was most reliable whenthe IPG P-type so obtained was resuspended in 100 μl water, of which 1or 2 μl were used for assay in the PDH phosphatase activation system.Under these conditions, a stimulation of up to +300% was obtained andthe response was linear within the parameters given. When amounts ofplacenta greater than 0.3 g were used, the yield of P-type IPG(calculated per gram extracted) fell sharply, either due to theco-extraction of a potent inhibitor or the presence of materialscompeting for the available binding sites on the resin.

No activity of IPG A-type, as evidenced by stimulation of lipogenesis inrat adipocytes, was detected. Six separate pH 1.3 extracts prepared fromplacenta; 4 separate extractions were tested, all in triplicate.Parallel extractions of rat liver, carried out at the same time, yieldedvalues of 1.92 units/g and an insulin standard assayed at the same timegave a stimulation above base-line of +258%.

5. Expression of Results

A unit of IPG is defined as the amount causing a 50% activation in thebasal level of the test system.

The yield of IPGS in urine is given on three different bases:

(i) Percentage stimulation of the test system by 10 μl final urineextract (Col 1), allowing direct comparison with data of Asplin et. al.[15]

(ii) Units of IPG per 1 mmol creatinine.

(iii) Units of IPC found in a sample of a 24 hour collection or urine;ie: the total daily output at that stage of gestation.

The results are given as means ± SEM and are evaluated either on thebasis of the corresponding paired sample, the selection of subjectsbeing matched for stage of gestations, parity and age, or on the basisof the Mann-Whitney ranking test for non-parametric data.

6. Design of Experiment

The ten pre-eclamptic and pregnant diabetic women studied were eachmatched with a normal pregnancy control subject for stage of gestation(±13 days), parity (0, 1-3, 4+) and age (±4 years). The gestationalrange was from 26 to 37 weeks for the pre-eclamptic group and from 31 to38 weeks for the diabetic pregnant group. Normal non-pregnant women ofreproductive age were included to allow evaluation of the effect ofpregnancy per se on urinary excretion of inositol phosphoglycans.

The changes in inositol phosphoglycans in pre-eclampsia were correlatedwith the severity of the condition.

Urine: Creatinine, urea. Na⁺, K⁺, Ca⁺ protein and volume / 24 hours.

Blood: Creatinine, aspartate transaminase (liver enzyme marker),platelet counts.

Biodata: Age, gestational age, parity, blood pressure, birth weight,placental weight.

B. Results

1. Stability of Inositol Phoshoglycans Isolated from the Placenta:

Material from the first placenta, freeze-clamped immediately on receiptfrom the delivery room, gave exceptionally high yields of IPG P-type, noactivity of IPG A-type was found.

The activity of the immediate sample from the second placenta wasappreciably less but, nevertheless, contained approximately 7 units ofactivity/g. Samples from this placenta stored at room temperature 1, 3or 5 hours were all devoid of IPG P-type activity. The sample stored onice for 1 hour had lost approximately half of its activity compared tothe immediate freeze-clamped sample, while those stored for 3 or 5 hoursboth yielded less than 1 unit of activity/g. It is concluded that IPGsare highly unstable in untreated tissue, even at 0° C.

2. Inositol Phosphoglycans in Placenta Delivered at Term:

The values for units/g tissue of extracted IPG P-type and IPG A-type areshow in Table 1 for the human placenta, human liver and rat liver. Theexceptionally high value for the IPG P-type in placenta is apparent.

The occurrence of a very high IPG P-type in placenta is in accord withthe known function of this putative insulin mediator on steroidogenesisin this tissue, and with the reported action of IPG P-type in activatingglycogen synthase phosphatase [16, 17]. Further, as a firstapproximation, it may be held that an increase in urinary IPG P-type inpregnancy, whether in normal pregnant, diabetic or pre-eclampticsubjects, originates in the placenta. Thus, measurements of theconcentration and 24-hour daily excretion of IPG P-type may be anindicator of placental production of this mediator.

A comparative study of pre-eclamptic and normal placental tissuedemonstrates a 2.7 fold increase in IPG P-type in the pre-eclampticplacenta (See Table 5).

The inability of the pH 1.3 fractions from all six placenta studied tostimulate lipogenesis in rat adipocytes may indicate a high degree oftissue specificity for the placental IPG A-type and a very specializedfunction for placental IPG A-type.

3. Inositol Phosphoglycans in Urine in Pre-eclampsia and Diabetes:

The concentration and total daily output of inositol phosphoglycans inthe urine of pre-eclamptic or pregnant diabetic and control subjects aregiven in Table 2. The results are given as the stimulation of thebioassay system produced by 10 μl of the IPG P-type (pH 2.0) or the IPGA-type (pH 1.3) fractions to allow comparison with the data of Asplinet. al. [15]. The results are also shown as units/mmol creatinine and asthe 24-hour daily output in units. The most striking difference was seenin the IPG P-type in the pre-eclamptic group which was two to three-foldhigher than the matched control group. Diabetes did not result in asignificant difference in the inositol phosphoglycans in urine relativeto their control group at the same stage of pregnancy. The individualvalues showing the range of values for pre-eclamptic, diabetic andcontrol groups are presented in FIGS. 1 A, B.

An interesting difference resides in the present observation that thenon-pregnant control group had a lower IPG P-type concentration andtotal daily excretion than the pregnant control groups for thepre-eclamptic and diabetic subjects (see Table 2). These differenceswere statistically significant.

The striking finding that the pre-eclamptic group alone showed asignificant increase of P-type amongst the pregnant women, and that theIPG P-type was lower in the non-pregnant group, provided evidence for alink between pre-eclampsia and the production of this inositolphosphoglycan.

That the increased urinary IPG P-type originates in the placenta isstrongly suggested by the present observation that all pregnant subjectshad a raised IPG P-type value relative to the non-pregnant controls andthat the placenta itself has an outstandingly high endogenousconcentration of IPG P-type (see Table 5).

If the higher excretion rate of IPG P-type in pre-eclamptic subject andtheir matched controls compared to normal non-pregnant subjects isequated to the contribution of the placenta to urinary IPG P-type (Table2), then the effect of pre-eclampsia is even more starkly underlinedwith the pre-eclamptic values being some 5- to 6- times greater than thepre-eclamptic control “control for the pre-eclamptic group” values forall modes of expression (Table 3) . This interpretation is strengthenedwhen a similar calculation is made for the IPG A-type. In this case,there is no ‘excess’ IPG A-type that can be ascribed to the presence ofthe placenta, a finding in accord with the present inability todemonstrate the presence of the A-type in this tissue (see Table 1)Also, values fall post-natally, as shown in FIG. 6.

The only significant difference in urinary IPG A-type recorded in Table2 and FIG. 1 C, D was the raised value in pre-eclamptic subjects whenthe results were expressed as units/mmole creatinine. No difference wasseen in the 24 hour output of IPG A-type in this condition.

4. Inositol Phosphoglycans in Non-pregnant Subjects:

The ten non-pregnant subjects in this study included five on thecontraceptive pill and in order to determine if the altered hormonebackground might influence the profile of inositol phosphoglycans, thesetwo sub-sets were considered separately. These data are shown in FIG. 2from which it can be seen that there is some evidence for a higheroutput of P-type in the group taking contraceptive pills. The number ofsubjects in each sub-set is too small on which to base firm conclusionsbut, nevertheless, the results suggest that an extension of this surveyis merited.

In a separate project, on the changes in urinary IPGs in male diabeticsubjects attending the out-patients clinic at The Middlesex Hospital, agroup of normal males was involved. Comparison of the data from thatstudy with those from the present study of normal non-pregnant femalesrevealed the interesting finding that while the IPG P-type/mmolcreatinine was similar in both groups, the IPG A-type was significantlyhigher in the normal female urine samples by 5- to 6-fold (Table 4).There was a marked difference in the IPG P-/IPG A- ratio, which was 0.6for women and 3.1 for men.

5. Inositol Phosphoglycans and Stage of Gestation:

In view of the evidence for a progressive decrease in the activity of anumber of placental enzymes involved in glucose metabolism and inplacental glycogen content towards the end of gestation [5, 18-20], thepresent data on urinary inositol phosphoglycans were examined withrespect to the stage of gestation at which the samples were taken; thisvaried between 26-37 weeks.

While these data must be interpreted with caution in view of the smallnumber of samples at the earlier stages of gestation and the possibleweighting by one remarkably high value at 26 weeks, certain trends areapparent.

There was a significant correlation between the stage of gestation andIPG P-type in the urine of pre-eclamptic subjects (r=0.609; P<0.05)(FIG. 3A), the highest values of IPG P-type being found at the beginningof the third trimester, the values for pre-eclamptic and age-matchedcontrol groups approximating closely in the period 35-37 weeks. No suchcorrelation was seen with the diabetic or normal control groups (seeFIG. 3B). Further, no correlation was found between IPG A-type in urineand the stage of gestation.

In this present study, single 24 hour urine collections were made atdifferent stages in the period 26-37 weeks of pregnancy. It is open tospeculation whether the differences between IPG P-type in the urine ofthe pre-eclamptic group and their matched controls reflect a relativeimmaturity of the cells in the pre-eclamptic subjects [4, 21], and adelay in a naturally occurring decline in IPG P-type production towardsthe end of gestation, or whether the significant raised values of IPGP-type in the pre-eclamptic group is a specific marker for the degree ofseverity of pre-eclampsia. The question of the possible changes in IPGP-type in urine at different stages of pregnancy remains to be answeredby measurements of urinary IPGs in the same subject at timed intervalsthroughout pregnancy.

6. Urinary Inositol Phosphoglycans and Markers for Pre-eclampsia:

6.1 IPG P-type and Markers of Pre-eclampsia:

The IPG P-type excreted was examined in relation to markers ofpre-eclampsia, including protein in urine, the activity ofalanine-aspartate transaminase in plasma and blood pressure, all knownto increase in pre-eclampsia, and platelet counts which decrease.

The correlations between these different markers and IPG P-type/mmolecreatinine in pre-eclamptic subjects are shown in FIGS. 4 A-C. Insummary these results show that:

(i) Protein in urine—positively correlated with IPG P-type, P<0.01.

(ii) Alanine—aspartate transaminase—positively correlated, P<0.05.

(iii) Platelet count—negatively correlated with IPG P-type, P<0.05.

6.2 IPG A-type and Markers of Pre-eclampsia:

It was noted, in Table 2, that IPG A-type showed an upward trend inpre-eclamptic subjects although this was only significant on the basisof IPG A-type/mmol creatinine and was less marked than the IPG P-type.While the present relatively small differences in a sample of tensubjects makes conclusions drawn from these results only tentative, itwas found that there was a positive correlation between increased IPGA-type and systolic blood pressure (P<0.05). This observation is,perhaps, strengthened by the parallel correlation found in a separatestudy of 31 diabetic male subjects in which there was a clear positivecorrelation between IPG A-type and raised blood pressure. However, it isprobable that rigorous control of blood pressure would be maintained inall pre-eclamptic subjects, masking possible underlying links betweenIPGs and blood pressure in the present study.

C. Discussion

The possibility that inositol phosphoglycans may be particularlyimportant as part of the signal transduction system in placenta inregulating glucose and glycogen metabolism and steroidogenesis stemsfrom the evidence that:

(i) Insulin mediators of this class can be isolated from placentalplasma membranes [22] and from freeze-clamped intact placental tissue(Table 1);

(ii) IPG P-type activates glycogen synthase phosphatase and pyruvatedehydrogenase phosphatase [6, 7, 11, 17];

(iii) Inositol phosphoglycans have been shown to be a signaltransduction system in the regulation of human placental steroidogenesis[16, 23].

The present results show clearly that the human placenta is aparticularly rich source of IPG P-type and differences betweennon-pregnant and pregnant groups provides strong evidence that the risein urinary content in normal pregnancy and in pre-eclampsia originatesfrom the placenta. This was confirmed by direct analysis of placentasamples. The highly significant two- to three-fold rise in IPG P-type inurine of the pre-eclamptic group, over and above the matched controlgroup, a difference that is even more significant at earlier stages ofpregnancy (FIG. 3A), has been shown to correlate with markers ofpre-eclampsia (FIGS. 4 A-C). Further, the elevated IPG P-type providesan explanation for the striking accumulation of glycogen in the placentain pre-eclampsia (Scheme 1).

It is postulated that different mechanisms are involved in the glycogenaccumulation in the placenta in pre-eclampsia and diabetes (see Scheme1). In contrast to pre-eclampsia, there is no evidence for a rise in IPGP-type in the urine in pregnant diabetic subjects relative to theirmatched controls (Table 2) and in this condition the increasedaccumulation of glycogen may relate to the increases in glucose andglucose 6-phosphate in this tissue as shown by Shafrir et. al [5, 24].As pointed out by these authors, both glucose and glucose 6-phosphateare activators of glycogen synthase, the raised accumulation of glycogenthus relates to the maternal hyperglycaemia. In this regard, theplacenta in diabetes shows certain parallelisms to the response of thekidney in diabetes, the latter has a raised glucose and glucose6-phosphate content and also accumulates glycogen. Thus, both tissuesexhibit features of glucose over-utilization in diabetes [25].

While the present results show a close association between markers ofpre-eclampsia and the concentration and daily excretion of IPG P-type wedo not know whether this is ‘cause’ or ‘effect’. It is likely that thecirculating concentration of IPG P- type in the plasma is also increasedin pre-eclampsia. Should experimental data confirm this assumption, thequestion can then be asked as to whether the raised circulating level ofa mediator, which can activate both paracrine and autocrine signallingsystems, might affect the functions of other tissues and endocrineorgans.

The exposure of endothelial cells, widely thought to be dysfunctional inpre-eclampsia [3], to raised levels of IPG P-type, might be critical inthe link between IPG P-type production in the placenta and systemiceffects. The inositol phosphoglycans appear to have autocrine andparacrine regulatory functions affecting placental steroidogenesis [16],insulin-dependent progesterone synthesis in swine ovary granulosa cells[23], FSH and HCG stimulus of granulosa cells [26]. ACTH signalling ofbovine adrenal cells [27], TSH stimulation of thyroid cells [28], IGF1stimulation of BALB/C3T3 granulosa cells [29], transforming growthfactor B on chondrocytes [30], and activation of human platelets [31].

A particularly interesting facet of the present study is the apparentabsence of extractable IPG A-type from six different placentae and frommultiple samples from a single placenta (Table 1). A number ofexplanations can be put forward. It may be that IPG A-type decreasesmarkedly in the third trimester, that it is less stable in the placentaafter birth and before freezing and extraction, that it has a hgihdegree of tissue specificity and is not active in the adipocyte assaysystem or, more interestingly, that it is, indeed, markedly low in theplacenta. In contradistinction to IPG P-type, the enzymes affected byIPG A-type include a number centering upon regulation of proteinphosphorylation, these include: inhibition of adenylate cyclase and cAMPdependent protein kinase and activation of a membrane bound low Km cAMPphosphodiesterase [6, 7].

It could be argued that placenta is essentially a unidirectional system,transferring nutrients from the maternal to the fetal circulation andacting as a buffering system for glucose in the storage and release ofglycogen. The on/off and the biosynthetic/degradation cycles regulatedby protein phosphorylation cycles seen in liver(glycolysis/gluconeogenesis) and adipose tissue (lipogenesis/lipolysis)do not appear to play a major role in placental function [18-20].Further work is needed in this aspect of regulation of placentalmetabolism better to define the regulation of the synthesis and the roleof inositol phosphoglycans.

The new findings of increased excretion of IPG P-type in urine, and thepossibility that it originates in the placenta in pre-eclampsia,suggests a mechanism for the accumulation of glycogen in thesyncytiotrophoblast of pre-eclamptic pregnancies. The correlation ofthese changes in IPG P-type with the markers of the severity of thesyndrome of pre-eclampsia also suggests that at least part of thisdysfunction may arise from excessive levels of the signalling system.

TABLE 1 Yields of IPG P-type and IPG A-type from placenta and liverCalculated Calculated Calculated Units/(g) Units/g Units/g IPG P-typeIPG P-type IPG P A-type Wt taken (g) (pH 2.0) Wt taken (g) (pH 2.0) (pH1.3) PRE- CONTROL ECLAMPTIC Human placenta Placenta No Placenta No PE1JB0.18 581 Con 8CA 0.34 266 ND 0.26 522 0.49 159 ND 0.47 242 1.05 47 ND1.03 81 Human Liver 1.82 1.60 (n = 2) Rat Liver 2.60 ± 0.22 2.6 ± 0.12(n = 13) ND—none detected

TABLE 2 The concentration and total daily output of inositolphosphoglycans in the urine of pre- eclamptic, or pregnant diabetic andmatched control subjects and a non-pregnant control group. IPG P-Type 24daily IPG A-Type 24 hour Stimulation Units/mmol output StimulationUnits/mmol daily output Creatinine 10 μl urine creatinine (units) 10 μlurine creatinine (units) (mmol/L) PRE-ECLAMPTIC GROUP CONTROLS 94.1 ±11.1  30.5 ± 8.94  316 ± 62   81.4 ± 8.4  25.9 ± 3.48  251 ± 42 6.94 ±0.90 (n = 10) PRE-ECLAMPSIA  205 ± 43**  96.4 ± 29.2** 854 ± 318**  102± 8.4  48.5 ± 6.98** 407 ± 78 4.79 ± 0.58 (n = 10) PREGNANT DIABETICGROUP CONTROLS  129 ± 26   38.5 ± 5.12  374 ± 47   84.5 ± 13.5 29.2 ±5.5   294 ± 57 6.41 ± 0.53 (n = 10) DIABETICS 89.8 ± 11.0  37.3 ± 7.64 275 ± 48   82.1 ± 9.4  34.6 ± 8.12  253 ± 45 5.94 ± 0.76 (n = 10)NON-PREGNANT GROUP (n = 10) 68.2 ± 12.3  18.8 ± 1.98  187 ± 25   97.8 ±11.8 32.7 ± 6.02  346 ± 75 7.11 ± 0.81 The results are given as Means ±SEM for 10 subjects in each group. The control subjects for thepre-eclamptic and diabetic groups were matched for gestational stage,parity and age; a normal non-pregnant group is shown. The data are givenas the percentage stimulation of pyruvate dehydrogenase phosphatase (IPGP-type) or percentage stimulation of lipogenesis (IPG A-type) (i) perunit volume of urine (10 μl = 10 ml urine); # (ii) as units of IPGactivity/mmol creatinine or (iii) as total 24 hour output of units ofIPG activity. 1 unit is defined as the amount of IPG causing a 50%increase in bioassay system. The statistical significance was assessedby the Mann-Whitney test *P, <0.05: **P, <0.01

TABLE 3 Calculation of the placental contribution to the amount of IPGP-type found in the urine of pregnant women, including diabetic,pre-eclamptic (PET) and their matched control groups. DIFFERENCESBETWEEN PREGNANT AND NON-PREGNANT IPG P-TYPE GROUPS GROUP (units/mmolcreatinine) Non-pregnant 18.8 ± 1.98 — group Pregnant groups Diabeticgroup Controls 38.5 ± 5.12 +19.7 ± 5.12 Diabetics 37.3 ± 7.64 +18.5 ±7.64 Ratio D/C 0.97 0.94 Pre-eclamptic group Controls 30.5 ± 8.94 +11.7± 8.94 Pre-eclampsia 96.4 ± 29.2 +77.6 ± 29.2 Ratio PET/C 3.2  6.6 

Calculated from data in Table 2. Each group contained 10 values; theresults are given as means ±SEM.

TABLE 4 The IPG P-type and IPG A-type content of urines from normalnon-pregnant female subjects and from normal male subjects. IPG P-typeIPG A-type IPG P Units/mmol creatinine IPA A Females (10) 18.8 ± 1.9832.7 ± 6.02 0.57 No treatment 16.1 ± 2.14 29.5 ± 6.02 (5) 21.5 ± 3.0143.4 ± 10.5 Males (27) 19.3 ± 1.8  6.30 ± 0.78 3.06 P value, female NS<0.001 v male

Values for males are taken from a separate survey of IPA A- and IPGP-types in the urine of diabetic and control male subjects.

TABLE 5 IPG P-type in human placenta and urine in pre- eclamptic andnormal pregnant subjects. Pre-eclamptic Control PE/C Placenta [1]Units/g Units/g IPG P-type 81 ± 11 (5) 30 ± 6 (4)** 2.7 Urine [2]Units/24 h Units/24 h IPG P-type 854 ± 318 (10) 316 ± 62 (10)** 2.7 [1]Non-matched samples of placenta [2] Urine samples matched forgestational age and parity IPG P-type activates pryuvate dehydrogenasephosphatase and glycogen synthase phosphatase. A unit of IPG P-typeactivity is defined as the amount producing a 50% stimulation of PDHphosphatase. Values are the means ± SEM; Fisher's P values shown by **P< 0.01.

TABLE 6 Placental Glycogen content & glycogen synthase activity [3].Pre- eclamptic Control PE/C Chorionic villi - glycogen content ug/gtissue) 1300 570 2.3 STB microvesicles - glycogen content (ug/mgprotein) 223 25 9.7 STB micro vesicles-glycogen synthase (units/mgprotein) 1323 83 16 [3] Data from: Arkwright, Rolemaker, Dwek, Redman1993) J Clin Invest 91:2744-2753

TABLE 7 IPG CONTENT OF PRE-ECLAMPTIC URINES ANTE-v POST-NATAL CreatinineUnits/L Units/mmol Total State mmol/L urine creatinine excretion IPGP-TYPE Ante- 6.58 ± 0.86 632 ± 112 98.9 ± 12.1 850 ± 105 Post- 5.78 ±0.64 406 ± 51  70.8 ± 12.2 747 ± 127 Paired t NS NS ** ** IPG A-TYPEAnte-  149 ± 23.2 26.2 ± 5.6  234 ± 60  Post-  148 ± 19.2 30.4 ± 6.5 286 ± 66  Paired t NS NS NS n = 9

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What is claimed is:
 1. A method of diagnosing pre-eclampsia in apatient, the method comprising determining the level of P-type IPGs in abiological sample obtained from the patient, wherein an elevated levelof P-type IPGs, as compared to a control level, is indicative ofpre-eclampsia or risk for pre-eclampsia, wherein the level of P-typeIPGs is determined using an assay selected from the group consisting of:(i) measurement of activation of pyruvate dehydrogenase phosphatase;(ii) measurement of activation of glycogen synthetase phosphatase; and(iii) an immunoassay.
 2. The method of claim 1 wherein the level of theP-type IPGs is determined using an assay measuring activation ofglycogen synthetase phosphatase by P-type IPGs.
 3. The method of claim 1wherein the level of the P-type IPGs is determined in an assay measuringactivation of pyruvate dehydrogenase phosphatase by P-type IPGs.
 4. Themethod of claim 1 wherein the level of the P-type IPGs is determinedusing an immunoassay.
 5. The method of claim 4, the method comprising:(a) contacting a biological sample obtained from the patient with asolid support having immobilized thereon antibody having one or morebinding sites specific for one or more P-type IPGs; (b) contacting thesolid support with a labelled developing agent capable of binding toP-type IPG's bound to antibodies or capable of binding to anti-P-typeIPG antibodies; and (c) detecting the label of the developing agentspecifically binding in (b) to obtain a value representative of thelevel of the P-type IPGs in the sample.
 6. The method of claim 1 whereinan elevated level of the P-type IPGs is greater than about 2 times thelevel in control subjects.
 7. The method of claim 1 wherein the sampleis a blood, serum, tissue or urine sample.
 8. The method of claim 2wherein an elevated level of the P-type IPGs is greater than about 2times the level in control subjects.
 9. The method of claim 3 wherein anelevated level of the P-type IPGs is greater than about 2 times thelevel in control subjects.
 10. The method of claim 4 wherein an elevatedlevel of the P-type IPGs is greater than about 2 times the level incontrol subjects.
 11. The method of claim 4 wherein the sample is ablood, serum, tissue or urine sample.
 12. The method of claim 5 whereinan elevated level of the P-type IPGs is greater than about 2 times thelevel in control subjects.
 13. The method of claim 5 wherein the sampleis a blood, serum, tissue or urine sample.
 14. The method of claim 5wherein the labelled developing agent is capable of binding to boundP-type IPGs.
 15. A method of diagnosing pre-eclampsia in a patient, themethod comprising determining the level of P-type IPGs in a biologicalsample obtained from the patient, wherein said P-type IPGs are capableof activating pyruvate dehydrogenase phosphatase, and a P-type IPG levelelevated by more than about 2-fold, as compared to a control level, isindicative of pre-eclampsia or risk for pre-eclampsia, wherein the levelof P-type IPGs is determined using an immunoassay.
 16. The method ofclaim 15 wherein the sample is a blood, serum, tissue or urine sample.17. The method of claim 16, the method comprising: (a) contacting thesample with a solid support having an anti-P-type IPG antibodyimmobilized thereon; (b) contacting the solid support with a labelleddeveloping agent capable of binding to P-type IPG's bound to antibodiesor capable of binding to anti-P-type IPG antibodies; and (c) detectingthe label of the developing agent specifically binding in (b) to obtaina value representative of the level of the P-type IPGs in the sample.18. The method of claim 17 wherein (i) the immobilized antibodycomprises a monoclonal antibody, (ii) after contact with the sample, thesolid support is contacted with a polyclonal antibody capable ofspecifically binding to IPGs, and (iii) the developing agent comprisesan antibody capable of specifically binding to the polyclonal antibody.